The covalent attachment of biologically active molecules to the surface of a substrate can be useful in a variety of applications such as in diagnostic devices, affinity separations, high throughput DNA sequencing applications, the clean-up of polymerase chain reactions (PCR), and the like. Immobilized biological amines, for example, can be used for the medical diagnosis of a disease or genetic defect or for detection of various biomolecules.
The modification of solid supports (e.g. particulate chromatography supports) by introduction of reactive functional groups for the immobilization of any of a variety of ligands is known. The attachment of a nucleophile (e.g., NH2, SH, OH, etc.) to a substrate may be achieved through the use of tethering compounds. A tethering compound has at least two reactive functional groups separated by a linking group. One of the functional groups provides a means for anchoring the tethering compound to a substrate or support by reacting with a complementary functional group on the surface of the substrate. A second reactive functional group can be selected to react with the nucleophile-containing material. Supports containing hydroxyl groups (e.g. cellulose, cross-linked dextrans, wool, and polyvinyl alcohol) may be treated with cyanuric chloride (trichlorotriazine) for the attachment of enzymes, antigens, and antibodies. Hydroxyl-containing supports such as Sepharose may be reacted with trichlorotriazine (TCT) which may then bind one or more nucleophiles. Solid nylon beads derivatized with cyanuric chloride have been used for oligonucleotide based hybridization assays. TCT coated paper and nylon membranes have also demonstrated utility in transfer hybridization experiments of DNA, RNA, and proteins.
Known tethering compounds are typically highly reactive with nucleophile-containing materials including biological materials. But, the reaction of the tethering compounds to nucleophile-containing materials may compete with other reactions, such as the hydrolysis of the tethering compound, when reactions with nucleophiles are conducted in aqueous solutions. Hydrolysis can result in incomplete or inefficient immobilization of the nucleophile-containing materials on a substrate.
There is a need for improved immobilization substrates and for tethering compounds compatible with such substrates. Accordingly, it is desired to provide supports and tethering compounds that are useful for ligand immobilization in any of a variety of applications.